223 Molecular Characterization of Myiasis-Causing Moth Flies

Home , Clogmia, Clogmia albipunctata, Psychodidae, Psychodinae, Telmatoscopus

Original Investigation / Özgün Araştırma 223

Molecular Characterization of Myiasis-Causing Moth Flies (Diptera: Psychodidae) Myiasis Nedeni Güve Sineklerinin (Diptera: Psychodidae) Moleküler Karakterizasyonu

Zuhal Önder , Abdullah İnci , Alparslan Yıldırım , Arif Çiloğlu , Önder Düzlü

Department of Parasitology, Erciyes University School of Medicine, Kayseri, Turkey

Cite this article as: Önder Z, İnci A, Yıldırım A, Çiloğlu A, Düzlü Ö. Molecular Characterization of Myiasis-Causing Moth Flies (Diptera: Psychodidae). Turkiye Parazitol Derg 2018; 42(3): 223-8.

ABSTRACT Objective: The aim of this study is to examine the molecular characterization of moth flies (Diptera: Psychodidae) based on their mitochondrial DNA sequences and determine the vectorial potential and damage caused by moth flies in future researches. Methods: A total of 240 adult moth flies were collected from toilet, bathroom, and basement walls of houses from different locations of the Kayseri region between May 2016 and April 2017. The polymerase chain reaction (PCR) analyses were performed using primer pairs, specificaly targeting the mitochondrial cytochrome oxidase c subunit I (mt-COI) gene of adult flies. Results: In total, five isolates were gel purified and sequenced for molecular characterization and phylogenetic analyses. Two species, namely Telma- toscopus albipunctatus (ERU-Telmatos3 and ERU-Telmatos6) and Psychodidae sp. (ERU-Psycho1,4,5), were successfully identified with the sequence alignment of isolates. According to the phylogenetic analysis, it was determined that the ERU-Telmatos3 and ERU-Telmatos6 isolates are clustered in the haplogroup A, while the ERU-Psycho1,4,5 isolate was clustered within the haplogroup B. The ERU-Psycho1 isolate was characterized as a new haplotype within the haplogroup B. Conclusion: This study represents the first molecular characterization and phylogenetic status of moth flies in Turkey. The obtained findings should be the first step in the future investigation based on detecting the transmission of bacterial pathogens by moth flies. Keywords: Moth fly, myiasis, molecular characterization, phylogenetic analysis Received: 16.03.2018 Accepted: 03.05.2018

ÖZ Amaç: Bu çalışma, mitokondrial DNA sekansı temelinde güve sineklerinin (Diptera: Psychodidae) moleküler karakterizasyonunu, gelecek araştırmalar- da bu sineklerin neden olduğu zararları ve vektörlük potansiyellerini belirlemek amacıyla yapılmıştır. Yöntemler: Mayıs 2016-Nisan 2017 yılları arasında Kayseri’nin çeşitli bölgelerinde konutların banyo, tuvalet ve bodrum duvarlarından toplam 240 ergin güve sineği toplanmıştr. Ergin sineklerin mitokondrial sitokrom oksidaz I (COI) gen bölgesini amplifiye eden spesifik primer çiftleri ile polimeraz zincir reaksiyonu (PCR) analizleri yapılmıştır. Bulgular: Toplam 5 izolat moleküler karakterizasyon ve filogenetik analizler için jel pürifiye edilip sekanslanmıştır. İzolatların sekans analizleri ile Telma- toscopus albipunctatus (ERU-Telmatos3 ve ERU-Telmatos6) ve Psychodidae sp. (ERU- Psycho1,4,5) türleri identifiye edilmiştir. Filogenetik analizlere göre ERU-Telmatos3 ve ERU-Telmatos6 izolatları haplogroup A içinde, ERU-Psycho1,4,5 izolatları ise haplogroup B içinde kümelenmiştir. Aynı zamanda ERU-Psycho1 izolatı haplogroup B’de yeni bir haplotip olarak karakterize edilmiştir. Sonuç: Bu çalışma, Türkiye’de güve sineklerinin moleküler karakterizasyonu ve filogenetik durumu üzerine ilk moleküler verileri sağlamıştır. Çalışma ile elde edilen sonuçlar, myiasis nedeni güve sineklerinin bakteriyel patojenlerin taşınması ve mekanik vektörlük potansiyellerinin belirlenmesi üzerine gelecek araştırmaların ilk basamağı olması bakımından oldukça orijinaldir. Anahtar Kelimeler: Güve sineği, myiasis, moleküler karakterizasyon, filogenetik analiz Geliş Tarihi: 16.03.2018 Kabul Tarihi: 03.05.2018

INTRODUCTION chodinae is commonly known as the drain, moth, or filter fly The Psychodidae family has a cosmopolitan distribution, because these are commonly found on the sites of buildings, with approximately 3000 described species in six subfamilies such as sewers, cesspools, septic tank, sewage treatment (Horaiellinae, Sycoracinae, Trichomyiinae, Bruchomyiinae, plants, and other dark and moist places (3). The females of Phlebotominae, and Psychodinae) (1, 2). The subfamily Psy- moth flies are not blood feeding, and they feed on flower

Address for Correspondence / Yazışma Adresi: Zuhal Önder E.mail: [email protected] DOI: 10.5152/tpd.2018.5943 ©Copyright 2018 Turkish Society for Parasitology - Available online at www.turkiyeparazitolderg.org ©Telif hakkı 2018 Türkiye Parazitoloji Derneği - Makale metnine www.turkiyeparazitolderg.org web sayfasından ulaşılabilir. Önder et al. Turkiye Parazitol Derg 224Molecular Characterization of Moth Flies 2018; 42(3): 223-8 nectar, sewage, and other moisture sources. Their larval stages Scientific, USA) according to the manufacturer’s instructions. The are coprophagous and saprophagous, and during these stages, extracted genomic DNA samples were stored at −20oC until the they feed on a gelatinous film of decaying organic matter and PCR analyses. vertebrate faeces (4-7). PCR Amplification Moth flies [Psychoda and Telmastoscopus (formerly Clogmia)] of Extracted DNA from each sample was used to amplify a 709 the Psychodinae subfamily are of public health and veterinary bp fragment of the mitochondrial COI gene barcoding region consideration (8-10). Both genera are commonly found on base- using the LCO-1490 forward (5’-GGTCAACAAATCATAAAGA- ment walls, in homes, and in public places such as restrooms TATTGG-3’) and HCO-2198 reverse (5’-TAAACTTCAGGGTGAC- and the bathrooms in sports centeres, hospitals, and hotels (11). CAAAAAATCA-3’) primers (20). The concentration and purity of When the hairs of these flies are inhaled, people cannot breathe extracted DNA was measured by a Qubit 3.0 fluorometer (Life or function comfortably, and this may even trigger respiratory Technologies). problems such as allergic rhinitis and asthma in humans (7). In addition, adult moth flies can be mechanical vectors of several The PCR was performed in a final reaction volume of 25 µL, con- bacterial pathogens associated with nosocomial infections. The taining 0.4 µM of each primer, 0.2 mM of each dNTP (Fermentas, moth fly larvae may cause accidental myiasis (e.g, urinary, intesti- Vilnius, Lithuania), 50 mM KCl, 10 Mm Tris-HCl (pH 9.0), 4 mM nal, and nasal myiasis) (4, 10, 12). MgCl2, 1U Taq DNA polymerase (Thermo Fisher Scientific, Hud- son, NH, USA), and 50 ng of template DNA. DNase-RNase-free Correct species identification of the moth fly is highly important water was used as negative control. The reaction was performed for pest control practices (13). According to external morpho- in a C1000 Touch™ thermal cycler (Bio-Rad, USA), and this com- logical characters, traditional morphological identification of the prısed an initial denaturation step at 94°C for 3 min, followed by Psychodinae species is difficult (14). 39 cycles of denaturation at 94°C for 20 sec, primer annealing at At present, several DNA-based analyses have been devel- 50°C for 20 sec, and a primer extension at 72°C for 30 sec. The oped for the specific identification and characterization of in- final extension at 72°C for 10 min was performed, and samples sect species. The mitochondrial DNA sequences have been were held at 4°C (17). The PCR products were electrophoresed successfully used for the identification of species in many dif- on a 1.5% agarose gel containing 10 μL/mL ethidium bromide in ferent insect groups (15, 16). Two of the mitochondrial genes, the Tris-acetate-EDTA (TAE) buffer at 135 V for 30 min and visual- cytochrome oxidase subunit I and II (COI and COII), are most ized using the CLP Gel Documentation System (UVP INC Upland, reliable and popular molecular markers. Cytochrome c ox- CA). idase subunit I (COI) has highly conserved sequences and Sequence Alignment and Phylogenetic and Genetic Distance variable regions within vertebrates and insects (15-17). An Analyses understanding of evolutionary relationships among different subfamilies and species of the psychodid flies, especially in The amplified PCR products were gel purified using the High the moth flies, are of epidemiological importance, because Pure PCR product purification kit (Roche, Mannheim, Germany) the phylogenetic relationship helps to predict the mechani- following the manufacturer’s instructions. In addition, purified cal transmission of bacterial pathogens such as Acinetobacter DNA was bidirectionally sequenced (Macrogen, Korea) with baumannii, Aeromonas hydrophila, Alcaligenes faecalis, Bacil- the same primers used to PCR amplify a 709 bp region of the lus cereus, Escherichia coli, Klebsiella pneumoniae ssp. pneu- mitochondrial COI gene. The nucleotide chromatograms were moniae, Pseudomonas aeruginosa, P. fluorescens, and Ste- aligned with the Geneious 9.1.3 software [www.geneious.com notrophomonas maltophili in houses, hospitals (bathrooms, (21)]. The DNA polymorphism, the number of haplotypes, and patient room toilets, and hospitals kitchens), and public toilets nucleotide diversity were determined using DnaSP 5.10.01 (22). and bathrooms (18, 19). Therefore, the aim of this study was Calculations for the mean genetic diversities within and among to determine the molecular characterization and phylogenetic the haplogroups were performed with the MEGA version 6 (23) status of the moth flies in Turkey. by the Kimura two-parameter distance model (24, 25). METHODS Phylogenetic analyses were performed by the Bayesian (BA) inference. The best-fit DNA-substitution model based on the Collection of Samples and Genomic DNA Isolation Akaike information criterion algorithm was determined as the The fly specimens were collected from toilet, bathroom, and General Time Reversible (GTR) by using jModeltest v.0.1.1 (26, basement walls of houses from different locations of the Kayseri 27). The BA analyses were set up in the MrBayes version 3.2.6 (28) region between May 2016 and April 2017 using suitable nets. and PhyML (29), respectively, by using the plugins available with The captured flies were placed in 1.5 mL eppendorf tubes con- the Geneious 9.1.3 software (www.geneious.com, 21). The spe- taining the 70% ethyl alcohol solution, and they were transported cies of moth flies in the Genbank used for phylogenetic analysis to the laboratory. are detailed in Table 1. The whole body of each sample was crushed in a 1.5 ml micro- The Markov chain simulations in the BA search were run for 10 centrifuge tube with liquid nitrogen in a pre-cooled mortar. The million generations, with sampling every 200 generations for the total DNA was extracted individually from each crushed fly us- posterior probability calculations. The 25% of the parameter es- ing the GeneJET Genomic DNA Purification Kit (Thermo Fisher timates was discarded as burn-in. Turkiye Parazitol Derg Önder et al. 2018; 42(3): 223-8 Molecular Characterization of Moth Flies225

Table 1. Species used from Genbank for sequence alignment

Genbank Accession No Species Isolate Origin AB907184 Telmatoscopus albipunctatus COI13 India HQ979025 Psychodidae sp. BOLD:AAF9305 USA KY924867 Telmatoscopus albipunctatus ERU-Telmatos3 Kayseri, Turkey KY924868 Telmatoscopus albipunctatus ERU-Telmatos3 Kayseri, Turkey KX054540 Psychodidae sp. sc_05918 French Polynesia KX054541 Psychodidae sp. sc_05919 French Polynesia HQ978770 Diptera sp. BOLD:AAE5173 Canada KR992085 Psychodidae gen. PschodididGC sp. BOLD-2016 Canada KR988152 Psychodidae gen. PschodididGC sp. BOLD-2016 Canada KR990808 Psychodidae gen. PschodididGC sp. 1 BOLD-2016 Canada KY924855 Psychodidae sp. ERU-Psycho4 Kayseri, Turkey KY924866 Psychodidae sp. ERU-Psycho5 Kayseri, Turkey KR990235 Psychodidae gen. PschodididGC sp. BOLD-2016 Canada KR770724 Psychodidae gen. PschodididGC sp. BOLD-2016 Canada KR722204 Psychodidae gen. PschodididGC sp. BOLD-2016 Canada KY924864 Psychodidae sp. ERU-Psycho1 Kayseri, Turkey

base composition was strongly AT biased for all species and sim- ilar to mitochondrial genes in insects (30); the mean GC content was 0.330. All the sequences were submitted to the NCBI-gene bank nucleotide database, and the accession number assigned was KY924864-68. Phylogenetic Analyses The BA tree is presented in Figure 2, with posterior probability (BA) and bootstrap support (ML) values. Two major haplogroups (A and B) and one new haplotype were found in this study. The isolates ERU-Telmatos3 and ERU-Telmatos6 were clustered Figure 1. Amplification of the mitochondrial COI gene in moth flies. Lane M: 100 bp DNA ladder; lane 1–7: PCR products of the with the isolates belonging to Telmatoscopus albipunctatus and COI gene (the samples isolated from different parts of Kayseri) Psychodidae sp., reported from India and the United States, , respectively, and they were placed under the haplogroup A1 RESULTS with a high bootstrap support (100%) and posterior probability COI Sequences and Genetic Divergence (1.00) (Figure 2). The mean genetic diversity among the isolates in the A1 haplogroup was 0.4±0.2%. A mean genetic distance A total of 240 adult flies were collected from 40 houses. A total of 3.5±0.8% was determined between the haplogroup A1 and of 50 genomic DNA isolates were amplified with the PCR and the haplotype A2, which clustered under the major group A. sequence analyses of the partial mt-COI gene region, and all the The isolates ERU-Psycho4 and ERU-Psycho5 showed a 100.0% isolates displayed a successful amplification (Figure 1). match with the isolates belonging to the Psychodidae sp. re- In total, five partial sequences (658 bp without primers) of moth ported from Canada and constituted the B2 haplogroup with flies specimens from the Kayseri region were obtained. Accord- the contribution of ERU-Psycho1 isolate, which was character- ing to sequence analyses of isolates, a total of two different ized as a new haplotype (Figure 2). This phylogenetic resolution moth flies (ERU-Telmatos3 and ERU-Telmatos6 isolates as Tel- was also supported with a moderate bootstrap support (58%) mastocopus albipunctatus and ERU-Psycho4, ERU-Psycho5, and and posterior probability (>0.56). The genetic difference be- ERU-Psycho1 as Psychodidae sp.) were identified. A total of 31 tween the ERU-Psycho1 isolate and other isolates under the B2 polymorphic sites were distributed among the COI sequences haplogroup was determined as 0.2±0.2%. The B1 haplogroup of the isolates, leading to the detection of three different hap- included the isolates of Diptera sp. and Psychodidae sp. report- lotypes (isolates ERU-Telmatos3 and ERU-Telmatos6 constituted ed from the United States and Canada, and it showed mean the first, ERU-Psycho4 and ERU-Psycho5 constituted the second, genetic distances of 0.5±0.2% to the isolates of the B2 hap- and ERU-Psycho1 constituted the third haplotypes). The mean logroup. The mean genetic distances between the group A and haplotype diversity among the sequences was 0.800±0.164. The group B were designated as 5.6±1.0%. Önder et al. Turkiye Parazitol Derg 226Molecular Characterization of Moth Flies 2018; 42(3): 223-8

Figure 2. Phylogenetic relationships among the obtained moth flies and some other moth fly isolates based on DNA barcoding. The evolutionary history was inferred by the Bayesian method based on the GTR+G substitution model using the Geneious 9.1.3 software. We used the bootstrap analysis, which was conducted with 1000 replicates by the MEGA6 software to estimate the node reliability of the trees. Sequences were given as the GenBank accession number and country. Sequences in red type were obtained in this study. The haplogroups are indicated with A and B. The Phlebotomus papatasi (GenBank Accession No. KR020561) mt-COI sequence was used as an outgroup

DISCUSSION been reported in toilets, kitchens, bathrooms, and sewers in Psychodinae are a subfamily of the Psychodidae family and different geographical regions of the world (10-12, 32). Adult C. known as moth, drain, or filter flies. Approximately 2000 species albipunctata was observed in kitchens, toilets, and bathrooms, within this subfamily have been described in about 100 genera and also in a compost heap in Belgium (5). Obona and Jezek distributed all over the world (31). Adult moth fly species have were collecting adult C. albipunctata in and on many buildings Turkiye Parazitol Derg Önder et al. 2018; 42(3): 223-8 Molecular Characterization of Moth Flies227 in different districts of Slovakia (14). Similarly, in this study, adult in the B1 haplogroup and ERU-Psycho1 was 0.5±0.2%. However, moth flies were collected from the walls of toilets, bathrooms, high genetic diversity (5.6±1.0%) was found between the groups and basements of buildings in Kayseri, Turkey. A and B. There are a limited number of studies on the phylogenetic relationship of the Psychodinae subfamily based on the Correct species identification of psychodid flies is important for mt-COI gene region in all world (18, 32). Espindola et al. (18) ex- pest control practices (13). The traditional morphological identi- amined phylogenetic relationships among 52 Palearctic taxa of fication of fly species used the morphological features between Psychodinae based on the mitochondrial DNA sequence. They species. Most authors suggested that the morphological characters of drain flies are most closely related to other insect species report monophyly of the subfamily Psychodinae, and they found (6, 14). However, the identification of adult psychodid species by Psychodini samples in Clade II and also Psychoda comprised in using morphological characters is difficult and can be misidenti- several subclades of Clade II. Mokhtar et al. (32) found larvae in a fied (33). Whereas phylogenetic relationships and classification 41-year-old female patient’s feces in Malaysia. They were exam- within the Psychodidae family is only known for the subfamily inining the larval specimen under a stereomicroscope and iden- Phlebotominae, species-rich Pscyhodinae subfamily relation- tified as a Psychodid larva. Then the COI gene region by DNA ships with other species and within the family remains poorly barcoding of larvae was amplified using primer pairs, and the understood (18). At present, studies on the clasification of the DNA barcode from the specimen suggested that the specimen Pscyhodinae subfamily are generally based on the morphology was the larval stage of C. albipunctatus. of all life stages (egg, larvae, and adult) and fossil species (34- CONCLUSION 37). Hennig (34) and Azar et al. (35) suggested a clade Tricho- myiinae+Sycoracinae+Horaiellinae to be the sister group of the Our study provided the first set of data on the molecular char- Psychodinae based on the morphology of mainly fossil species. acterization and phylogeny of moth flies based on the DNA barcoding in Turkey. We provide an understanding of phylogenetic With the development of molecular techniques, the PCR tech- reconstruction of these species to determine the vectorial po- nique, which mainly targets the nuclear and mitochondrial genes, tentiality and epidemiology of moth flies in Turkey, and thus the has been most commonly used in molecular characterization and results of our analyses may be fruitful in future research. Further- phylogenetic analyses of insect species in recent years (16, 17). more, this study may be helpful to determine proper pest man- The cytochrome b (cytb), mt-COI and mt-COII, NADH dehydro- agement practices against the moth flies. genase subunit 1, and 28S and 16S ribosomal RNA gene regions were used to determine relationships within the subfamily Psy- chodinae in different region of the world (18). The molecular Ethics Committee Approval: Due to the material composed from insect analyses of mitochondrial DNA have ensured diagnostic markers specimens Ethics Committee Approval was not needed for this study. for the identification of species in many different insect groups. The mitochondrial-encoded genes, COI and COII, are among Informed Consent: Not required in this study. the more robust and reliable molecular markers for identifica- Peer-review: Externally peer-reviewed. tion and phylogenetic analyses in most insect species because Author Contributions: Concept - Z.Ö., A.İ., A.Y.; Design - Z.Ö., A.İ., A.Y., this gene region has highly conserved sequences and variable Ö.D., A.Ç.; Supervision - Z.Ö., A.İ., A.Y.; Funding - Z.Ö., A.İ., A.Y.; Mate- regions in vertebrates and insects (15-17, 38). rials - Z.Ö., A.İ., A.Y.; Data Collection and/or Processing - Z.Ö., A.İ., A.Y., There have been no molecular-based studies conducted on Ö.D., A.Ç.; Analysis and/or Interpretation - Z.Ö., A.İ., A.Y.; Literature Re- moth flies in Turkey up to date, to the best of our knowledge. view - Z.Ö., A.İ., A.Y., Ö.D., A.Ç.; Writing - Z.Ö., A.İ., A.Y.; Critical Review In this study, we describe the genotype characterization of moth - Z.Ö., A.İ., A.Y. flies by the DNA barcoding method based on mt-COI. We fur- Conflict of Interest: Authors have no conflicts of interest to declare. ther determined phylogenetic relationships within the moth fly’s Financial Disclosure: The authors declared that this study has received specimens. Our results demonstrate that ERU-Telmatos3 and no financial support. ERU-Telmatos6 isolates are grouped with T. albipunctatus isolates in India and Psychodidae sp. isolate in the United States. These species are located under the A1 haplogroup with a high Etik Komite Onayı: Çalışma materyalini insektler oluşturduğundan etik bootstrap support (100%) and posterior probability (1.00) (Figure komite onayı alınmamıştır. 2). According to the sequence BLAST analyses, the mean genetic Hasta Onamı: Bu çalışma için hasta onamına gerek yoktur. distance between the A1 haplogroup and the A2 haplotype was Peer-review: Externally peer-reviewed. 3.5±0.8%. Yazar Katkıları: Fikir - Z.Ö., A.İ., A.Y.; Tasarım - Z.Ö., A.İ., A.Y., Ö.D., A.Ç.; Phylogenetic analyses revealed that the ERU-Psycho4 and Denetleme - Z.Ö., A.İ., A.Y.; Kaynaklar - Z.Ö., A.İ., A.Y.; Malzemeler - Z.Ö., ERU-Psycho5 isolates showed a 100% identity among the Psy- A.İ., A.Y.; Veri toplanmasıve/veya işlemesi - Z.Ö., A.İ., A.Y., Ö.D., A.Ç.; chodidae sp. isolates in Canada. The ERU-Psycho1 isolate within Analiz ve/veya yorum - Z.Ö., A.İ., A.Y.; Literatür taraması - Z.Ö., A.İ., A.Y., the B2 haplogroup was characterized as a new haplotype in this Ö.D., A.Ç.; Yazıyı yazan - Z.Ö., A.İ., A.Y.; Eleştirel İnceleme - Z.Ö., A.İ., A.Y. study. The genetic difference between the B2 haplogroup isolates and the ERU-Psycho1 isolates was calculated as 0.2±0.2%. Çıkar Çatışması: Yazarlar çıkar çatışması bildirmemişlerdir. The mean genetic distance between the Diptera sp. isolate from Finansal Destek: Yazarlar bu çalışma için finansal destek almadıklarını the United States and Psychodidae sp. isolate from Canada with- beyan etmişlerdir. Önder et al. Turkiye Parazitol Derg 228Molecular Characterization of Moth Flies 2018; 42(3): 223-8

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